December 1, 2015 (Vol. 35, No. 21)
Frank P. Elsen Ph.D. Electrophysiology Application Scientist Harvard Bioscience
Patch-Clamp Technology Allows Precise Investigation of Neural Mechanisms
Understanding how nerve cells (neurons) function is essential to finding medical cures for many neuronal diseases, such as epilepsy, Alzheimer’s, Parkinson’s, stroke, or even severe headaches. Since the days of Luigi Galvani (1737–1798), who used simple metal wires and clamps to investigate the mechanisms that made frog muscles twitch, neuroscience research has come a long way and scientists have started to use more and more advanced techniques. Nowadays researchers employ much more sophisticated equipment to uncover the mysteries of the brain.
The human brain has 100 billion neurons that form a complex network with approximately 1 trillion (1012) connections (synapses). To put these numbers in perspective, consider that the Milky Way has 100 billion stars and 1.1 trillion seconds ago, Neanderthals still roamed on earth. Given these huge numbers of neurons and synapses, it comes as no surprise that research on the mammalian brain is far from being complete. Listening in on the “conversations” between neurons would be a good first step to uncover the mechanism on how they “talk” to each other and to find out what is wrong, when neurons start to “scream” like during an epileptic seizure.
Today we know that neurons use electricity to communicate with each other. To “speak” and “listen” to a neuron, we need to establish a physical connection (patch-clamp technique) with them and then we can use their “words” (voltage, current, and resistance, etc.) and “grammar” (Ohm’s law, etc.) to communicate.
Developed by a group of researchers around Bert Sakmann and Erwin Neher (Göttingen/Germany 1976), the patch-clamp technique enables researchers to “chat” with neurons. It creates a connection between the neuron and the researcher, a pathway on which electric signals can travel back and forth. Neurons are able to generate tiny electrical signals (action potentials), because they express ion channels in their membrane.
Using those channels, ions (charged atoms) travel between the inside and outside of the cell (current), creating a potential difference (voltage) that travels as an action potential along the nerve fiber (axon) to the synapse, thereby sending a “message” to the next neuron. The first step in the connection pathway is to establish a very tight, physical connection with the membrane of the neuron. This is accomplished with a tiny glass capillary (patch pipette). Given the small size (5 to 20 µm, [10-6 m]) of cortical neurons (human hair diameter: 100 µm), the tip of the patch pipette needs to be even smaller (1 µm). Using special pipette pullers (like the HEKA PIP-6), researchers need to fabricate patch pipettes freshly each day.
Patch pipettes are filled with a special, salty solution and are placed in a pipette holder that connects to a pre-amplifier (headstage). It contains a key electronic element of the patch-clamp technique: the negative feedback circuitry. The main amplifier (HEKA EPC-10 USB) and the headstage together form the most important electronic components of the patch-clamp technique. They receive and amplify small analog signals from the neuron, transfer them into digital ones (with the help of an analog/digital converter inside the amplifier), and relay them to the computer. Reversing this pathway, they also convey “questions” from the researcher to the neuron. The headstage and amplifier are the central components: the turnstile or communication hub between the researcher and the neuron (Figure 1).
The electrical signals from a neuron are in the order of pico-Ampere (10-12 A, [pA]) and milli-Volts (10-3 V, [mV]). This is about a million times smaller than the current that would flow through the human body (1 to 100 mA) if you touched the electrical outlet (110 V) in your home. Modern-day amplifiers, like the EPC-10 USB have all the necessary components (AD/DA converter) incorporated into them. This, together with carefully constructed setups, enables scientists to separate the small cellular signals from electrical background noise. To measure these tiny electrical signals, specialized recording equipment is needed.
Neurons need to be put inside specially created and carefully controlled environments (setup or rig) that protect them from any electric noise, comparable to sound-proofed telephone booth that would cancel out background noise and thereby help you to listen to very low-volume conversations.
These instruments are controlled by PatchMaster (from Harvard Bioscience), a sophisticated acquisition software. The communication (current or voltage traces) from the neurons is displayed and recorded and in addition, the researcher can easily analyze the recorded data by using specialized build-in analysis functions.
How to Establish the Connection to the Neuron
To establish a physical connection to the neuron (patching the cell), the researcher has to perform a set of complex tasks that need to be exactly choreographed. One task is moving the pipette tip close to the cell membrane, while monitoring the approach on a 400 times magnified live picture. The EPC-10 USB patch clamp amplifier sends out a voltage signal that helps the user to determine when the tip makes contact with the cell membrane.
The voltage signal then creates a current that flows out of the tip of the pipette. The moment the tip makes contact with the membrane, the opening gets obstructed (pipette resistance increases), causing the current amplitude to decrease significantly. The researcher observes this amplitude reduction together with the optical observation of the tip touching the membrane (dip-forming) and must react accordingly (applying instant negative pressure to the interior of the pipette) to establish a tight connection between the pipette tip and the cell membrane (a so-called Giga-Ohm seal).
Depending on what recording configuration the researcher needs for the experiment, further physical treatments (changing pressure inside the pipette and/or moving pipette away from cell), together with electrical observations have to be performed manually by the researcher.
This whole procedure is very tricky and difficult to perform, therefore it is desirable to automate it. The only automatic patch-clamp machine that performs a pipette-based approach is the PatchServer from Multi Channel Systems. It allows the user to automatically establish whole-cell as well as single-channel recording configurations using any kind of cell type, as long as the cells are in suspension (Figure 2). Moreover, the PatchServer can be combined with an EPC-10 USB Quadro amplifier and then be used in four-channel recording mode. This enables the user to acquire data from four individual neurons at the same time, thereby significantly increasing the production rate (Figure 3).
Patch-clamp amplifiers like the HEKA EPC-10 USB enable researchers to investigate the cellular mechanisms of neurons. Using the patch-clamp technique gives us an idea of how neurons generate those small electrical signals, and how they are used for the communication among one another. Understanding the underlying mechanisms will help to uncover new neuronal pathways and mechanisms. We will be able to even better understand how neurons function and what could be wrong, if they malfunction. Ultimately, we hope that this will lead to the discovery of new cures for many diseases.
High-quality research equipment will lead to better data and thereby more precise analysis and conclusions. Easier to use (automatic), highly reliable patch-clamp equipment will give researchers a better chance to find the answers needed to understand the functionality of the mammalian brain. The human brain is an incredible organ. Let’s use it!